Lucas R. Chadwick

The addition of hops to fermented beer in the presence of yeast may result in a secondary fermentation commonly referred to as “after-fermentation,” “dry-hop creep,” “ABV creep,” and “the freshening power of the hop” (FPH). The effect is most likely caused by glycolytic enzyme(s) in hops converting residual nonfermentable polysaccharides from malt into simple sugars that are then fermented by yeast. The FPH must be considered because the ethanol increase can exceed ±0.3% ABV tolerance set out in the U.S. Code of Federal Regulations. Furthermore, with unpasteurized and unfiltered beers, the commensurate rise in carbonation levels may lead to high pressures and potential consumer hazard. Development of a quantitative measurement system for FPH will be a critical component in an ongoing challenge in the industry to consistently ‘brew to final gravity’ while minimizing handling and blending prior to packaging and to ensure ABV compliance. The research presented herein was carried out to characterize the FPH of Centennial hops, identify key factors involved, compare the magnitude of hop-induced secondary fermentation in common dry-hopping processes, and provide a roadmap to develop a quantitative protocol to predict FPH potential in any given sample of hops. In this paper, we review the current understanding of FPH and present a relatively simple and affordable method to measure FPH impact to better understand and control ABV, CO₂ output, and diacetyl in beers that are dry-hopped in the presence of live yeast and brewed to final gravity.

Countercurrent separation (CCS) utilizes the differential partitioning behavior of analytes between two immiscible liquid phases. We introduce the first platform (“CherryOne”) capable of real-time monitoring, metering, and control of the dynamic liquid–liquid CCS process. Automated phase monitoring and volumetrics are made possible with an array of sensors, including the new permittivity-based phase metering apparatus (PMA). Volumetric data for each liquid phase are converted into a dynamic real-time display of stationary phase retention (Sf) and eluent partition coefficients (K), which represent critical parameters of CCS reproducibility. When coupled with the elution–extrusion operational mode (EECCC), automated Sf and K determination empowers untargeted and targeted applications ranging from metabolomic analysis to preparative purifications.

The present study demonstrates the importance of adequate precision when reporting the δ and J parameters of frequency domain ¹H NMR (HNMR) data. Using a variety of structural classes (terpenoids, phenolics, alkaloids) from different taxa (plants, cyanobacteria), this study develops rationales that explain the importance of enhanced precision in NMR spectroscopic analysis and rationalizes the need for reporting Δδ and ΔJ values at the 0.1–1 ppb and 10 mHz level, respectively. Spectral simulations paired with iteration are shown to be essential tools for complete spectral interpretation, adequate precision, and unambiguous HNMR-driven dereplication and metabolomic analysis. The broader applicability of the recommendation relates to the physicochemical properties of hydrogen (¹H) and its ubiquity in organic molecules, making HNMR spectra an integral component of structure elucidation and verification. Regardless of origin or molecular weight, the HNMR spectrum of a compound can be very complex and encode a wealth of structural information that is often obscured by limited spectral dispersion and the occurrence of higher order effects. This altogether limits spectral interpretation, confines decoding of the underlying spin parameters, and explains the major challenge associated with the translation of HNMR spectra into tabulated information. On the other hand, the reproducibility of the spectral data set of any (new) chemical entity is essential for its structure elucidation and subsequent dereplication. Handling and documenting HNMR data with adequate precision is critical for establishing unequivocal links between chemical structure, analytical data, metabolomes, and biological activity. Using the full potential of HNMR spectra will facilitate the general reproducibility for future studies of bioactive chemicals, especially of compounds obtained from the diversity of terrestrial and marine organisms.

Inhibitors of quinone reductase-2 (NQO2; QR-2) can have antimalarial activity and antitumor activities or can function as chemoprevention agents by preventing the metabolic activation of toxic quinones such as menadione. To expedite the search for new natural product inhibitors of QR-2, we developed a screening assay based on ultrafiltration liquid chromatography−mass spectrometry that is compatible with complex samples such as bacterial or botanical extracts. Human QR-2 was prepared recombinantly, and the known QR-2 inhibitor, resveratrol, was used as a positive control and as a competitive ligand to eliminate false positives. Ultrafiltration LC−MS screening of extracts of marine sediment bacteria resulted in the discovery of tetrangulol methyl ether as an inhibitor of QR-2. When applied to the screening of hop extracts from the botanical, Humulus lupulus L., xanthohumol and xanthohumol D were identified as ligands of QR-2. Inhibition of QR-2 by these ligands was confirmed using a functional enzyme assay. Furthermore, binding of xanthohumol and xanthohumol D to the active site of QR-2 was confirmed using X-ray crystallography. Ultrafiltration LC−MS was shown to be a useful assay for the discovery of inhibitors of QR-2 in complex matrixes such as extracts of bacteria and botanicals.

Used in the brewing of beer, hops (Humulus lupulus L.) contain the prenylated chalcone xanthohumol, which is under investigation as a cancer chemoprevention agent and as a precursor for the estrogenic flavanones isoxanthohumol and 8‐prenylnaringenin. The uptake, transport and accumulation of xanthohumol were studied using the human intestinal epithelial cell line Caco‐2 to help understand the poor bioavailability of this chalcone. Studies were carried out using Caco‐2 cell monolayers 18–21 days after seeding. The apparent K_m and V_max values of xanthohumol accumulation in Caco‐2 cells were determined, and the protein binding of xanthohumol in sub‐cellular fractions of Caco‐2 cells was investigated. Approximately 70% of xanthohumol added to the apical side of Caco‐2 cells accumulated inside the cells, while 93% of the intracellular xanthohumol was localized in the cytosol. Xanthohumol accumulation was temperature dependent and saturable with an apparent K_m value of 26.5 ± 4.66 μM and an apparent V_max of 0.215 ± 0.018 nmol/mg protein/min. Facilitated transport was not responsible for the uptake of xanthohumol, instead, accumulation inside the Caco‐2 cells was apparently the result of specific binding to cytosolic proteins. These data suggest that specific binding of xanthohumol to cytosolic proteins in intestinal epithelial cells contributes to the poor oral bioavailability observed previously in vivo.

The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with K_m values of 14.8 ± 3.2 and 16.6 ± 4.6 μM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a K_m of 3.7 ± 0.9 μM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a K_m value of 17.8 ± 3.7 μM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.

Purpose The absorption potential and metabolism of 8-prenylnaringenin (8-PN) from hops (Humulus lupulus L.) were investigated. 8-PN is a potent estrogen with the potential to be used for the relief of menopausal symptoms in women. Methods Monolayers of the human intestinal epithelial cancer cell line Caco-2 and human hepatocytes were incubated with 8-PN to model its intestinal absorption and hepatic metabolism, respectively. Results The apparent permeability coefficients for 8-PN in the apical-to-basolateral and basolateral-to-apical directions of a Caco-2 monolayer were 5.2 ± 0.7 × 10⁻⁵ and 4.9 ± 0.5 × 10⁻⁵ cm/s, respectively, indicating good intestinal absorption via passive diffusion. Both glucuronide and sulfate conjugates of 8-PN were detected in the Caco-2 cell incubations. The 4′-O-glucuronide was the predominant Caco-2 cell metabolite, followed by 7-O-sulfate and 4′-O-sulfate. Both phase I and phase II metabolites of 8-PN were formed by human hepatocytes. The 7-O-glucuronide was the most abundant hepatocyte metabolite, and no sulfate conjugates were detected. Incubations with various cDNA-expressed UDP-glucuronosyltransferases indicated that the isozymes UGT1A1, UGT1A6, UGT1A8, and UGT1A9 were responsible for glucuronidation of 8-PN. Conclusions Although orally administered 8-PN should be readily absorbed from the intestine, its bioavailability should be reduced significantly by intestinal and hepatic metabolism.

The gram-negative bacterium Helicobacter pylori (HP), identified in 1982, is now recognized as the primary etiological factor associated with the development of gastritis and peptic ulcer disease. In addition, HP infections are also associated with chronic gastritis, gastric carcinoma and primary gastric B-cell lymphoma. For centuries, herbals have been used in traditional medicine to treat a wide range of ailments, including gastrointestinal (GI) disorders such as dyspepsia, gastritis and peptic ulcer disease (PUD). However, the mechanism of action by which these botanicals exert their therapeutic effects has not been completely elucidated. As part of an ongoing screening program, the study assessed the in vitro susceptibility of 15 HP strains to botanical extracts, which have a history of traditional use in the treatment of GI disorders. Methanol extracts of Myristica fragrans (seed) had a MIC of 12.5 µg/mL; Zingiber officinale (ginger rhizome/root) and Rosmarinus officinalis (rosemary leaf) had an MIC of 25 µg/mL. Methanol extracts of botanicals with a MIC of 50 µg/mL included Achillea millefolium, Foeniculum vulgare (seed), Passiflora incarnata (herb), Origanum majorana (herb) and a (1:1) combination of Curcuma longa (root) and ginger rhizome. Botanical extracts with a MIC of 100 µg/mL included Carum carvi (seed), Elettaria cardamomum (seed), Gentiana lutea (roots), Juniper communis (berry), Lavandula angustifolia (flowers), Melissa officinalis (leaves), Mentha piperita (leaves) and Pimpinella anisum (seed). Methanol extracts of Matricaria recutita (flowers) and Ginkgo biloba (leaves) had a MIC > 100 µg/mL. Copyright © 2005 John Wiley & Sons, Ltd.